Prostate cancer is the most common malignancy in males in Europe and North America (52). In the United States, more than 200,000 men develop prostate cancer each year, and more than 30,000 men die from prostate cancer each year. In initial stages, prostate cancers are localized and androgen-dependent; in advanced stages, they are invasive, metastatic, and heterogeneous in terms of androgen-dependence. The standard treatment for advanced prostate cancer is androgen ablation. Such treatment induces the apoptosis of androgen-dependent cells and reduces the size of primary and metastatic lesions; however, the surviving androgen-resistant cells typically form aggressive, drug-resistant populations. These observations emphasize the need for effective treatments for advanced prostate cancer.
The purine analogs roscovitine, R-roscovitine (CYC002), and CGP74514A inhibit the activity of cyclin-dependent kinases (CDKs), most notably cdk2, cdk1, and cdk7 (1-3). Roscovitine is the compound 6-benzylamino-2-[(R)-1-ethyl-2-hydroxye-thylamino]-9-isopropylpurine. R-roscovitine refers to the R enantiomer of roscovitine, specifically the compound 2-(1-R-hydroxymethylpropylamino)-6-benzylamino-9-iso-propylpurine. CGP74514A is the compound N-(cis-2-Aminocyclohexyl)-N-(3-chlorophenyl)-9-ethyl-9H-purine-2,6-diamine. cdk2 and cdk1 promote the entry of cells into S phase and mitosis, respectively, and cdk7 facilitates transcription. These inhibitors have been linked to apoptosis, though the mechanisms are unclear (2, 4-9).
Apoptosis requires the activation of a family of cysteine aspartyl proteases termed the caspases (10). Adaptor proteins promote the autocleavage and activation of initiator caspases (e.g., caspase-8 and caspase-9), initiator caspases cleave and activate effector caspases (e.g., caspase-3), and effector caspases induce a multiplicity of events that ultimately result in cell death (e.g., plasma membrane blebbing and DNA fragmentation). There are two apoptosis signaling pathways: the death receptor pathway and the mitochondrial pathway. When active, death receptors interact with adaptor proteins such as FADD and TRADD and activate caspase-8. When damaged, mitochondria release cytochrome c, which associates with the adaptor protein Apaf-1 and activates caspase-9. Most drugs signal apoptosis through the mitochondrial pathway.
Proteins that modulate caspase activity—and thus determine whether cells live or die—include the IAPs (Inhibitor of Apoptosis Proteins) and the Bcl-2 proteins. The IAP family includes cIAP-1, cIAP-2, XIAP, and survivin (11). Of these proteins, XIAP is the most potent. IAPs interact with and inhibit the activity of processed caspases; thus, they function as ‘brakes’ that can impede the apoptotic process once it begins. IAPs inactivate both initiator and effector caspases; caspase-9 and caspase-3 are IAP targets, whereas caspase-8 is not (12).
The Bcl-2 proteins are critical determinants of mitochondria-dependent caspase activation (13). Some Bcl-2 proteins are apoptotic (e.g., Bax, Bak, and the BH3-only proteins), whereas others are anti-apoptotic (e.g., Bcl-2 and Bcl-XL). Death stimuli activate Bax and Bak, which perforate the outer mitochondrial membrane in a manner dependent on the BH3-only proteins. Although incompletely resolved, Bcl-2 and Bcl-XL prevent the activation of Bax and Bak by sequestering the BH3-only proteins or by interacting with Bax and Bak.
p53 is a transcription factor that often couples apoptotic signals to changes in the abundance and/or activity of the Bcl-2 proteins (14). It accumulates in cells in response to many chemotoxic drugs, typically as a result of stabilization, and it promotes apoptosis by both transcription-dependent and -independent mechanisms. In the nucleus, p53 transactivates genes encoding apoptotic proteins such as Bax and the BH3-only proteins Noxa and Puma (15-17). When localized to mitochondria, p53 activates Bax and Bak by interacting with Bcl-2 family members (18). The mitochondrial actions of p53 are newly described and incompletely characterized. Given its role as a death signal, it is not surprising that p53 is frequently mutated in human tumors (19).